Figure 1.
Amino acid starvation increases mitochondrial respiration and membrane potential.
HEK cells were cultured in 25(HG), 5 mM glucose (LG), or 5 mM galactose (Gal), with (+) or without (−) the 15 amino acids (AA) present in the standard formulation of DMEM (Table S1). The same notation is used in subsequent figures. Mitochondrial oxygen consumption rate (OCR) was measured using a flux analyser before (basal) (A) and after the addition of FCCP (maximal) (B), having subtracted the non-mitochondrial (rotenone-insensitive) OCR. Inset shows maximal OCR of HEK cells grown in the presence or absence of amino acids combining the three sets of values for the different sugar concentrations shown in panel B. (C) Mitochondrial membrane potential was assessed by a quantitative flow cytometry analysis of TMRE fluorescence. The fluorescence values were normalized to those of HEK cells grown in HG with amino acids. The data represent the mean ± standard error of the mean (s.e.m) of 3 independent experiments, each one performed in duplicate. Statistical analysis was performed using the unpaired two-tailed Student’s t-test. Asterisks indicate the level of statistical significance (P<0.05 *, P<0.01 **, and P<0.001 ***); NS, not significant (p>0.05).
Figure 2.
Amino acid starvation boosts mitochondrial protein synthesis.
(A) One-hour 35S-methionine pulse-labeling of nascent mitochondrial polypeptides in HEK cells grown for 72 h in different media (see Figure legend 1), and fractionated by 12% SDS-PAGE. Tentative assignments of the mitochondrial polypeptides are indicated to the left of the gel: COI-III, subunits of cytochrome c oxidase; ND1-6 and 4L, subunits of respiratory complex I; Cyt b – Cytochrome b of respiratory complex III; A6 and A8, subunits of ATP synthase. A section of the Coomassie blue-stained gel indicates equal protein loading. (B) Pulse-labelings of nascent mitochondrial polypeptides and fractionation as per panel A except that the labeling medium lacked all amino acids (other than 35S-methionine). (C) The combined signal of the labeled mitochondrial proteins was quantified and normalized with respect to HG+AA. HG, n = 6 experiments; LG, n = 3 experiments; and Gal, n = 3 experiments. The error bars represent the s.e.m.; unpaired student’s t-test, (P<0.05 *, P<0.01 **, and P<0.001 ***). (D) The growth rates of cells grown in HG with or without AA were monitored and measured over the course of 7 days. Broken vertical lines at 6, 26, and 72 h indicate the times at which the mitochondrial translation capacity was measured.
Figure 3.
Amino acid starvation does not induce mitochondrial biogenesis.
After growing HEK cells for 72: Respiratory complex I subunits, ND1, NDUSF1, and NDUFA7; complex II, succinate dehydrogenase subunit B; complex III, CORE2; complex IV, COVb and COII. TOM20, outer membrane protein of mitochondria (OM); GAPDH, reference protein. m, protein product of mtDNA; n, product of nuclear DNA.
Figure 4.
Amino acid deprivation increases the levels of several mitochondrial RNAs, their protein products, and TFAM and EFTu.
DNA, RNA, and protein were harvested from HEK cells grown for 72(A) and (B) Northern hybridization with probes corresponding to mRNAs and tRNAs, respectively. COIII, cytochrome oxidase III mRNA; ND1-3, RNAs encoding subunits of complex I; pRNA, precursor RNA; EB, ethidium bromide. 18S and 28S rRNA are the RNA elements of cytosolic ribosomes. R – arginine, Y – tryptophan, L(UUR) – Leucine. (C) Immunoblots of selected mitochondrial translation machinery factors: EFTUm – mitochondrial translation elongation factor; TFB1M, modifies the RNA of the small subunit of the mitochondrial ribosome; CRIF1, a contributor to normal mitochondrial translation; LRPPRC, a mitochondrial mRNA-interacting protein. (D) and (E) Immunoblots of TFAM and TFB2M, respectively. (F) Q-PCR estimation of mtDNA copy number; n = 6; error bars represent the s.e.m. There was no significant change in mtDNA copy number as a result of amino acid withdrawal based on the unpaired student’s t-test, NS not significant (p>0.05), for any sugar concentration, or all (inset). Using the same test there was a significant increase in the mtDNA copy number when glucose was replaced by galactose (Figure S6).
Figure 5.
HEK cells starved of amino acids have elevated levels of amino acid-catabolizing and TCA cycle enzymes and YY1, while displaying the signature of TORC1 inhibition.
HEK cell extracts were immunoblotted for (A) proteins involved in mitochondrial amino acid metabolism (DBT, GCSH, ASNS, see text for details) and aconitase 2. The chart shows the citrate synthase enzyme activity in the cell lysates (nmol citrate/s/mg protein). Error bars represent the s.e.m.; n = 3 (pairs); two asterisks, p<0.01. (B) The abundance of sensors and effectors linked to nutrient signaling in the different growth regimes was determined by immunoblotting for AMPK, 4E-BP1, S6K, and YY1, with GAPDH as reference. The reference corresponding to the blots of S6K and YY1 is not shown. The numbers following the amino acids indicate the key phosphorylated residues for which the antibody is specific. (C) schemes illustrating the influence of mitochondria on cellular anabolism (i) and catabolism (ii) according to amino acid availability. i) to proliferate cells require amino acids from the breakdown of food, in these conditions TORC1 is active; it promotes cytosolic protein synthesis (CPS) and inhibits protein recycling, mitochondria make relatively little contribution to energy production (which is more reliant on glycolysis – not shown). Conversely, amino acid deprivation (ii) inhibits TORC1, which leads to the repression of cytosolic protein synthesis (CPS) and the upregulation of mitochondrial protein synthesis (MPS) and the respiratory electron transport chain (RETC). YY1 also contributes to increased mitochondrial respiration, either independently as illustrated, or possibly via derepression owing to TORC1 inhibition.